Crystallin synthesis in lens differentiation in cultures of neural retinal cells of chick embryos.

Dissociated cells of neural retinas of 3.5-day-old chick embryos differentiated into “lentoid bodies” within about 10–12 days when cultured in vitro. Protein synthesis of these cultured cells was studied with the use of SDS-polyacrylamide gel electrophoresis, affinity chromatography, and autoradiography combined with immunological techniques. Incorporation of [¹⁴C]leucine into total proteins, α-crystallin, and δ-crystallin was estimated after increasing times of culture up to about 30 days. Isotope incorporation into δ-crystallin was detected at 9 days, and it increased sevenfold after another 17 days. α-Crystallin was also first detected at 9 days, but its relative content reached a maximum at 12 days and then decreased gradually. The ratio of δ-crystallin synthesis to total protein synthesis increased up to 40% at 26 days, while that of α-crystallin synthesis remained 3% throughout the culture period. These results show that lens differentiation from neural retinal cells is associated with the preferential synthesis of lens crystallins, particularly of δ-crystallin.     

Studies on the differentiation capacity of neural epithelium cells in chick embryos

Cell and Tissue Research -     

Differentiation of lens in cultures of neural retinal cells of chick embryos

When dissociated cells of neural retinae of 8-day-old chick embryos were cultured, monolayer sheets of epithelial cells were obtained. These cells proliferated actively. After about 30 days of culture, both lentoid bodies and pigment cells were differentiated in all plates. In the second and the third generation cultures, both differentiations were also observed. Lentoid bodies showed positive immunofluorescence for fluorescein-isothiocyanate-conjugated antiserum against δ-crystallin. Molecular constituents of lentoid bodies were very similar to those of lenses developing in situ, as revealed by immunodiffusion tests. Several lines of evidence for the “neural retinal” origin of lentoid bodies, as opposed to their being derived from lens cells inadvertently included in the original culture inocula are given. Some implications of the present results for the problem of “determination” are discussed.     

Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells of chick embryos.

When dissociated cells of neural retinae of 9-day-old chick embryos were cultured in Eagle's minimum essential medium supplemented with dialyzed fetal calf serum, both the proliferation and differentiation of the neural retinal cells were inhibited. These cells remained quiescent and flattened. When ascorbic acid was added to such a medium, the cells started to grow and differentiated into lentoid bodies and pigmented cells after about 10 days.     

Differentiation of lens and pigment cells in cultures of neural retinal cells of early chick embryos

Dissociated cells of neural retinas of 3.5-day-old chick embryos (stages 20–21) were cultured as a monolayer in order to examine their differentiation in vitro. These cells started to grow actively soon after inoculation and formed a confluent sheet within which neuroblast-like cells with long cytoplasmic processes were differentiated by 8 days. At about 16 days the differentiation of both lentoid bodies and foci of pigment cells was observed, while neuronal structure disappeared. The numbers of lentoid bodies and foci of pigmented cells continued to increase up to 30 days, when primary cultures were terminated. The increase in δ-crystallin content, as measured by quantitative immunoelectrophoresis assay using rabbit antiserum against δ-crystallin, was consistent with the increase in the number of lentoid bodies in cultures. The amount of α-crystallin per culture, estimated by the same technique as above, reached a maximum at 16 days and decreased slightly during further culture. The differentiation of both lentoid bodies and pigment cells was observed also in cultures of the second generation. The results demonstrate that cells of the undifferentiated neuroepithelium of 3.5-day-old embryonic retinas can achieve at least three differentiations, neuronal, lens, and pigment cells, in vitro. We discuss several differences between the present results and the previous ones from in vitro cultures of 8- to 9-day-old embryonic neural retinas.     

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-β1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.     

Effects of alkaline phosphatase inhibitors on chick neural retinal cell differentiation in vitro: ultracytochemical studies

In the matured chick retina, alkaline phosphatase (ALPase) activity is specifically localized in the outer plexiform layer and in horizontal and Müller cells. In the developing chick retina, ALPase activity is first recognized in growing neurites from horizontal cells during the 13th day of incubation, when synaptogenesis begins in the outer plexiform layer. Intraocular administration of ALPase inhibitors to developing chick embryos resulted in developmental disturbances in differentiation of the outer plexiform layer and also of photoreceptor cells. We have now extended these studies to an in vitro system. ALPase activity was studied by ultracytochemistry in cultured retinal cells from chick embryos, and the effects of specific ALPase inhibitor on retinal development were also analyzed. Two cell types showed intense ALPase activity: 1) flat glial cell, which formed a multi-layered epithelial sheet and 2) neuronal cell found within cell aggregates. Some cellular processes forming a neuropil-like structure within these aggregates also showed ALPase activity. When the ALPase inhibitor bromotetramisole was present in the culture medium, there was delay in aggregate formation and the development of neuritic processes was also affected. Moreover, this treatment also caused a considerable reduction in the number of photoreceptor cells present in the culture. The present results indicate that ALPase activity plays a significant role in retinal cell differentiation.     

In vitro analysis of sympathetic neuron differentiation from chick neural crest cells

Sympathetic neuron differentiation was studied using a fluorescence histochemical assay to detect the appearance of cell-bound catecholamines. Results from in vitro organ cultures indicate that chick neural crest cells must interact with both ventral neural tube (defined throughout as the ventral neural tube plus the notochord) and somitic mesenchyme in order to differentiate into sympathoblasts. Somite, ventral neural tube, and crest were cultured transfilter in various combinations to define these tissue interactions more precisely. Results from these experiments indicate that neural crest cells must be contiguous to somite in order to differentiate into sympathoblasts, but ventral neural tube may act across a Millipore filter membrane (type TH, 25 μm thick) either on somite, crest, or both. To distinguish among these possibilities, somite was cultured transfilter to ventral tube for a short period, after which ventral tube was removed and fresh crest was added to the somite. The results from this and other experiments support the hypothesis that the ventral tube does not act directly on crest cells, but elicits a developmental change in somitic mesenchyme, which then promotes sympathoblast differentiation. To study the relationship of nerve growth factor (NGF) to the differentiation of sympathetic neurons, cultures of somite + crest were temporarily exposed transfilter to ventral tube, in the presence or the absence of exogenous NGF. The results of these and other experiments are consistent with the hypothesis that the continued presence of ventral tube is required to ensure the survival of the differentiating sympathetic neurons. With respect to this second function, ventral tube can be replaced by exogenous NGF.     

Nerve growth factor induces neural differentiation in undifferentiated cell of early chick embryos

Nerve growth factor (NGF) induced differentiation in postnodal pieces (PNPs) of stage 4 chick embryos. This induction was highly selective for neural tissue; no other structures developed in the NGF-treated PNPs. Furthermore, the number of PNPs showing neural differentiation was dependent on the concentration of NGF, but there was no correlation between the concentration of NGF (5-100 ng/ml) and extent of neuralization. The neural inducing capacity of NGF could be abolished by anti-NGF antibody. NGF-induced neural differentiation was accompanied by elevated intracellular levels of cyclic AMP. Exogenous cyclic AMP (175 micrograms/ml) was able to stimulate neural differentiation but, unlike NGF, induced other structures (e.g., notochord and pulsatile tissue). Overall results suggest that cells from chick embryos at developmental stages much earlier than previously thought are responsive to NGF and NGF or a a closely related substance may serve as a neural inducer in the chick embryo.     

Morphometric analysis of neural tube cells in neurulating chick embryos]

Preliminary report on a morphometric study of the neuroepithelium of 1 S and 7 S -- stage chick embryos. We show first that the nucleo-cytoplasmic ratio and then, that the volume fraction of intercellular spaces, nuclei and cytoplasm as they relate to the neural tube, differ significatively from cephalic to caudal portion of the neural tube. At stage 7 S only the volume fraction of intercellular spaces, nuclei and cytoplasm in the neural tube change along the cephalo-caudal axis of the embryos. Finally, between the two stages, at the back of the head, 1) volume fraction of intercellular spaces in the neural tube remains unchanged 2) nucleo-cytoplasmic ratio increase 3) volumetric density and size of mitochondria do not change, while surface density of endoplasmic reticulum in cytoplasm increases.     

Silver staining of DNA synthesizing cells in the neural tube of chick embryos

Abstract. I report a study by light microscopy of the spinal cord of early chick embryos stained with the ammoniacal silver carbonate solution of Del Rio Hortega. Cell nuclei are stained in a selective fashion and two classes of nuclei – dark and pale – can be distinguished in the neuroepithelium. Neuronal nuclei also show a characteristic staining pattern. A radioautographic study after [³H] tyhmidine incorporation has shown that it is the dark neuroepithelial nuclei that are engaged in DNA synthesis. Dark nuclei disappear after administration of cytosine arabinoside, supporting the association between DNA synthesis and silver staining of neuroepithelial nuclei.     

Silver staining of DNA synthesizing cells in the neural tube of chick embryos

I report a study by light microscopy of the spinal cord of early chick embryos stained with the ammoniacal silver carbonate solution of Del Rio Hortega. Cell nuclei are stained in a selective fashion and two classes of nuclei - dark and pale - can be distinguished in the neuroepithelium. Neuronal nuclei also show a characteristic staining pattern. A radioautographic study after [3H] thymidine incorporation has shown that it is the dark neuroepithelial nuclei that are engaged in DNA synthesis. Dark nuclei disappear after administration of cytosine arabinoside, supporting the association between DNA synthesis and silver staining of neuroepithelial nuclei.     

Quantification of ganglioside GM1 synthetase activity on intact chick neural retinal cells

Neural retinal cells from 9-d-old chick embryos were assayed for uridine diphosphate (UDP)-galactose:ganglioside GM2 galactosyltransferase, or GM1 synthetase, activity using the oligosaccharide fragment of GM2, oligo-GM2, oligo-GM2, as the exogenous acceptor. The results demonstrated that this enzyme activity was present on the external surfaces of intact cells. Little difference between the specific activities of cell surface GM1 synthetase could be detected when cells derived from dorsal and ventral segments of the neural retina were compared. These results suggested that this cell- surface enzyme was not present in a concentration gradient along the dorsoventral axis of the neural retina.     

Lens differentiation in cultures of neural retinal cells of human fetuses

Neural retinal cells of human fetuses at approximately 9 and 15 weeks after conception were cultured in vitro. In early stages of culturing (up to about 10 days), a number of neuronal cells with axon-like processes were formed. Within about 20 days, many neuronal cells started to degenerate, while a number of “lentoid bodies” were identified by immunoelectrophoresis and immunofluorescent techniques using anti-rat lens serum which cross-reacted with human crystallins. Ultrastructural observations also revealed that cells of “lentoid bodies” represent typical profiles of lens fibers.     

Neuronal differentiation of mouse neural crest cells in vitro

The purpose of the present study is to analyze the effect of serum or chick embryo extract (CEE) on the neuronal differentiation of the mouse neural crest cells. When the crest cells were cultured in the medium containing serum at low concentration (5% calf serum), neurite outgrowth was observed. The active outgrowth was detected at 3-4 days in culture. However, in the medium supplemented with 20% calf serum, no neurite appeared, and the crest cells remained fibroblast-like. The differentiation of adrenergic neurons was observed when the crest cells were cultured in the medium containing CEE along with serum.     

In vitro differentiation of retinal pigment epithelium from adult retinal stem cells

One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.